Stress resistant plants

ABSTRACT

Stress tolerance in plants and plant cells is achieved by using nucleotide sequences encoding enzymes involved in the NAD salvage synthesis pathway and/or the NAD de novo synthesis pathway from fungal or yeast like organisms other than  Saccharomyces cereviseae , e.g., for overexpression in plants.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage filing of InternationalApplication No. PCT/EP2007/002433, filed Mar. 16, 2007, which claimspriority to EP 06075671.5, filed Mar. 21, 2006; U.S. Provisional PatentApplication No. 60/784,179, filed Mar. 21, 2006; and EP 06075700.2,filed Mar. 22, 2006, the disclosures of each of which are herebyincorporated by reference.

Methods are provided for increasing the stress resistance in plants andplant cells whereby enzymes involved in the NAD salvage synthesispathway and/or the NAD de novo synthesis pathway originating from fungalorganisms or yeasts, other than Saccharomyces cereviseae, are expressedin plants.

BACKGROUND ART

Tolerance of plants to adverse growing conditions, including drought,high light intensities, high temperatures, nutrient limitations, salinegrowing conditions and the like, is a very desired property for cropplants, in view of the never-ending quest to ultimately increase theactual yield of these plants.

Various ways of achieving that goal of improving what is commonly knownas the stress resistance or stress tolerance of plants have beendescribed. Since different abiotic stress conditions frequently resultin the generation of harmful reactive oxygen species (“ROS”) such assuperoxides or hydrogen peroxides, initial attempts to improve stressresistance in plants focused on prevention of the generation of the ROSor the removal thereof. Examples of these approaches are overexpressionof ROS scavenging enzymes such as catalases, peroxidases, superoxidedismutases etc. or even increasing the amount of ROS scavengingmolecules such as ascorbic acid, glutathione etc. These approaches andother attempts to engineer stress tolerant plants are reviewed e.g. inWang et al. 2003, Planta 218:1-14.

Stress tolerance in plant cells and plants can also be achieved byreducing the activity or the level of the endogenous poly-ADP-ribosepolymerases (ParP) or poly(ADP-ribose) glycohydrolases (ParG) asdescribed in WO00/04173 and PCT/EP2004/003995, respectively. It isthought that in this way, fatal NAD and ATP depletion in plant cellssubject to stress conditions, resulting in traumatic cell death, can beavoided or sufficiently postponed for the stressed cells to survive andacclimate to the stress conditions.

Uchimiya et al. (2002) et al. describe the isolation of a rice genedenoted YK1, as well as use of a chimeric YK1 gene to increase thetolerance of transgenic rice plants harboring that gene to rice blastand several abiotic stresses such as NaCl, UV-C, submergence, andhydrogen peroxide. (Uchimiya et al., 2002, Molecular breeding 9: 25-31).

Uchimiya et al. further published a poster abstract describing thatoverexpression of a NAD dependent reductase gene (YK1) in rice cellsalso promoted the level of NAD(P)(H) through up-regulating NADsynthetase activities, and concluded that this modification in turngenerated a pool of redox substances needed for ROS stress resistance(Uchimiya et al. 2003 Keystone symposium on Plant biology: Functions andcontrol of cell death, Snowbird Utah Apr. 10-15, 2003).

NAD synthetase from yeast has been well characterized and is the lastenzyme in both the NAD de novo synthesis pathway and the NAD salvage. Inthe de novo pathway, quinolate is the precursor for NAD synthesis and isgenerated as a product of tryptophan degradation. In the salvagepathway, nicotinamide (which is a degradation product of NAD, generatedthrough the action of various enzymes such as PARP, NAD-dependentdeacetylases or other NAD glycohydrolases) is the precursor molecule. Ina first step, nicotinamide is deamidated to nicotinic acid by anicotinamidase. The nicotinic acid is transferred to5-phosphoribosyl-1-pyrophosphate by the enzyme nicotinate phosphoribosyltransferase to yield nicotinic acid mononucleotide. This compound isshared between the de novo and the salvage pathway. Hence, furtherconversion of this compound by NAD+ pyrophosphorylase and NAD synthetaseis achieved as in the de novo pathway.

In yeast, overexpression of PNC1 (encoding nicotinamidase) has beencorrelated with life span extension by calorie restriction andlow-intensity stress (Anderson et al., 2003 Nature 423: p181-185; Galloet al., 2004, Molecular and Cellular Biology 24: 1301-1312).

WO2004/016726 describes methods and compositions for modulating the lifespan of eukaryotic and prokaryotic cells and for protecting cellsagainst certain stresses. One method comprises modulating the flux ofthe NAD+ salvage pathway in the cell, e.g. by modulating the level oractivity of one or more proteins selected from the group consisting ofPNC1, NMA1, NPT1 and NMA2.

Little is known about the respective enzymes of the NAD biosynthesispathways in plants. Hunt et al., 2004 describe the use of the availablegenomic information from Arabidopsis to identify the plant homologues ofthese enzymes (Hunt et al., 2004, New Phytologist163(1); 31-44). Theidentified DNA sequences have the following Accession numbers: fornicotinamidase: At5g23220; At5g23230 and At3g16190; for nicotinatephosphoribosyltransferase: At4g36940, At2g23420, for nicotinic acidmononucleotide adenyltransferase: At5g55810 and for NAD synthetase:At1g55090 (all nucleotide sequences are incorporated herein byreference).

PCT/EP 2005/010168 describes methods for increasing the stressresistance in plants and plant cells whereby enzymes involved in the NADsalvage synthesis pathway and/or the NAD de novo synthesis pathway areexpressed in plants.

Alternative methods for increasing stress tolerance in plants are stillrequired and the embodiments described hereinafter, including theclaims, provide such methods and means.

SUMMARY OF THE INVENTION

In one embodiment of the invention, a method is provided for obtaining aplant with increased stress resistance comprising introducing a chimericgene into a cells of a plant to obtain transgenic cells whereby thechimeric gene comprises the following operably linked DNA fragments:

-   -   i. A plant-expressible promoter;    -   ii. A DNA region coding for a plant-functional enzyme of the        nicotinamide adenine dinucleotide salvage synthesis pathway        selected from nicotinamidase, nicotinate        phosphoribosyltransferase, nicotinic acid mononucleotide adenyl        transferase or nicotinamide adenine dinucleotide synthetase    -   iii. A 3′end region involved in transcription termination and        polyadenylation,        followed by regenerating the transgenic cells to obtain a        population of transgenic plants; and selecting a plant from the        population of transgenic plants which exhibits increased stress        resistance or selecting a plant which exhibits a reduced level        of reactive oxygen species or maintains a high level of NADH        under stress conditions when compared to a similar        non-transgenic plant wherein said method is characterized in        that the amino acid sequence of the plant-functional enzyme        encoded by the DNA region comprises one of the following: the        amino acid sequence of accession number XP_(—)444840 (Candida        glabrata), the amino acid sequence of accession number        XP_(—)456073 (Kluyveromyces lactis), the amino acid sequence of        accession number NP_(—)986013 (Eremothecium gossypii), the amino        acid sequence of accession number XP_(—)888958 (Candida        albicans), the amino acid sequence of accession number XP500320        (Yarrowia lipolytica), the amino acid sequence of accession        number XP389372 (Giberella zeae), the amino acid sequence of        accession number XP_(—)749509 (Aspergillus fumigatus), the amino        acid sequence of accession number XP_(—)712112 (Candida        albicans), the amino acid sequence of accession number BAE56421        (Aspergillus oryzae), the amino acid sequence of accession        number XP_(—)567125 (Cryptococcus neofomans), the amino acid        sequence of accession number XP_(—)964547 (Neurospora crassa),        the amino acid sequence of accession number XP_(—)712135        (Candida albicans), the amino acid sequence of accession number        XP_(—)448179 (Candida glabrata), the amino acid sequence of        accession number XP_(—)453643 (Kluyveromyces lactis), the amino        acid sequence of accession number NP_(—)987024 (Eremothecium        gossypii), the amino acid sequence of accession number        XP_(—)500272 (Yarrowia lipolytica), the amino acid sequence of        accession number XP_(—)722371 (Candida albicans), the amino acid        sequence of accession number XP_(—)456405 (Debaromyces        hansenii), the amino acid sequence of accession number BAE61562        (Aspergillus oryzae), the amino acid sequence of accession        number XP_(—)759702 (Ustilago maydis), the amino acid sequence        of accession number EAL18D79 (Cryptococcus neoformans), the        amino acid sequence of accession number NP_(—)587771        (Schizosaccharomyces pombe), the amino acid sequence of        accession number XP_(—)681472 (Aspergillus nidulans), the amino        acid sequence of accession number XP_(—)959191 (Neurospora        crassa), the amino acid sequence of accession number        XP_(—)567726 (Cryptococcus neoformans), the amino acid sequence        of accession number EAQ90706 (Chaetomium globosum), the amino        acid sequence of accession number XP_(—)387574 (Giberella zeae),        the amino acid sequence of accession number XP_(—)748008        (Aspergillus fumigatus), the amino acid sequence of accession        number XP_(—)361704 (Magnaporthe grisea), the amino acid        sequence of accession number Q06178, the amino acid sequence of        accession number XP_(—)444815 (Candida glabrata), the amino acid        sequence of accession number NP_(—)986687 ((Eremothecium        gossypii), the amino acid sequence of accession number        XP_(—)453005 (Kluyveromyces lactis), the amino acid sequence of        accession number XP_(—)458184(Debaromyces hansenii), the amino        acid sequence of accession number XP_(—)718656 (Candida        albicans), the amino acid sequence of accession number        XP_(—)504391 (Yarrowia lipolytica), the amino acid sequence of        accession number NP_(—)592856 (Schizosaccharomyces pombe), the        amino acid sequence of accession number XP_(—)762639 (Ustilago        maydis), the amino acid sequence of accession number        XP_(—)571297 (Cryptococcus neoformans), the amino acid sequence        of accession number BAE57070 (Aspergillus oryzae), the amino        acid sequence of accession number XP_(—)750776 (Aspergillus        fumigatus), the amino acid sequence of accession number        XP_(—)659349 (Aspergillus nidulans), the amino acid sequence of        accession number XP_(—)389652 (Giberelia zeae), the amino acid        sequence of accession number XP_(—)957634 (Neurospora crassa),        the amino acid sequence of accession number XP_(—)363364        (Magnaporthe grisea), the amino acid sequence of accession        number XP_(—)758179 (Ustilago maydis), the amino acid sequence        of accession number EAQ85219 ((Chaetomium globosum), the amino        acid sequence of accession number CAA85352 (Saccharomyces        cerevisae), the amino acid sequence of accession number        XP_(—)448893 (Candida glabrata), the amino acid sequence of        accession number XP_(—)453357 (Kluyveromyces lactis), the amino        acid sequence of accession number NP_(—)983562 (Eremothecium        gossypii), the amino acid sequence of accession number        XP_(—)462577 (Debaromyces hansenii), the amino acid sequence of        accession number XP_(—)889008 (Candida albicans), the amino acid        sequence of accession number XP_(—)500338 (Yarrowia lipolytica),        the amino acid sequence of accession number XP_(—)746744        (Aspergillus fumigatus), the amino acid sequence of accession        number BAE64333 (Aspergillus oryzae), the amino acid sequence of        accession number XP_(—)965789 (Neurospora crassa), the amino        acid sequence of accession number EAQ93453 (Chaetomium        globosum), the amino acid sequence of accession number        XP_(—)682385 (Aspergillus nidulans), the amino acid sequence of        accession number AAN74808 (Gibberella moniliformis), the amino        acid sequence of accession number Q9UTK3, the amino acid        sequence of accession number XP_(—)361075 (Magnaporthe grisea),        the amino acid sequence of accession number EAL18922        (Cryptococcus neoformans), the amino acid sequence of accession        number XP_(—)568039 (Cryptococcus neoformans), the amino acid        sequence of accession number XP_(—)760597 (Ustilago maydis), the        amino acid sequence of accession number NP_(—)011524, the amino        acid sequence of accession number XP_(—)444815 (Candida        glabrata), the amino acid sequence of accession number        NP_(—)986687 ((Eremothecium gossypii), the amino acid sequence        of accession number XP_(—)453005 (Kluyveromyces lactis), the        amino acid sequence of accession number XP_(—)458184        (Debaromyces hansenii), the amino acid sequence of accession        number XP_(—)718656 (Candida albicans), the amino acid sequence        of accession number XP_(—)504391 (Yarrowia lipolytica), the        amino acid sequence of accession number NP_(—)592856        (Schizosaccharomyces pombe), the amino acid sequence of        accession number XP_(—)762639 (Ustilago maydis), the amino acid        sequence of accession number XP_(—)571297 (Cryptococcus        neoformans), the amino acid sequence of accession number        BAE57070 (Aspergillus oryzae), the amino acid sequence of        accession number XP_(—)750776 (Aspergillus fumigatus), the amino        acid sequence of accession number XP_(—)659349 (Aspergillus        nidulans), the amino acid sequence of accession number        XP_(—)389652 (Giberella zeae), the amino acid sequence of        accession number XP_(—)957634 (Neurospora crassa), the amino        acid sequence of accession number XP_(—)363364 (Magnaporthe        grisea), the amino acid sequence of accession number        XP_(—)758179 (Ustilago maydis) or the amino acid sequence of        accession number EAQ85219 (Chaetomium globosum).

In another embodiment, the invention relates to the chimeric genes asdescribed herein, plant cells comprising these chimeric genes, andplants consisting essentially of plant cells comprising these chimericgenes, and seeds of such plants. These plants and plant cells may becharacterized in that they have a lower level of reactive oxygen speciesunder stress conditions than a similar plant not comprising such achimeric gene.

In yet another embodiment, the invention relates to the use of thedescribed chimeric genes to increase the stress resistance of a plant orto decrease the level of reactive oxygen species in a plant or a plantcell under stress conditions.

The invention further provides the use of one of the mentioned DNAsequence encoding a plant functional enzyme of the nicotinamide adeninedinucleotide salvage synthesis pathway selected from nicotinamidase,nicotinate phosphoribosyltransferase, nicotinic acid mononucleotideadenyl transferase or nicotinamide adenine dinucleotide synthetase whichare from fungal or yeast-like origin to increase the stress resistanceof a plant or to decrease the level of reactive oxygen species ormaintain the level of NADH in a plant or a plant cell under stressconditions.

DETAILED DESCRIPTION

The current invention is based on the finding that DNA sequencesencoding plant-functional enzymes from the NAD salvage pathway in yeastscould be used to obtain transgenic plants which were more resistant tostress, particularly abiotic stress, than plants not comprising theseDNA sequences. The transgenic plants also exhibited a significantlyreduced level of reactive oxygen species (“ROS”) and maintained a highlevel of NADH, when put under stress conditions, compared to controlplants

Thus in one embodiment of the invention, a method is provided to obtaina plant with increased stress resistance, whereby the method comprisesthe steps of

-   -   introducing a stress resistant chimeric gene as herein described        into cells of a plant to obtain cells comprising the stress        resistant chimeric gene;    -   regenerating these cells comprising the stress resistant        chimeric gene to obtain a population of plants comprising the        stress resistant chimeric gene; and    -   selecting a plant from the population of these plants which        exhibits increased stress resistance and/or decreased ROS level        under stress conditions and/or maintains a high level of NADH,        when compared to a similar non-transgenic plant.

The stress resistant chimeric gene thereby comprises a plant-expressiblepromoter operably linked to a DNA region coding for a plant-functionalenzyme of the nicotinamide adenine dinucleotide salvage synthesispathway selected from nicotinamidase, nicotinatephosphoribosyltransferase, nicotinic acid mononucleotide adenyltransferase or nicotinamide adenine dinucleotide synthetase from fungalor yeast like origin and a 3′end region involved in transcriptiontermination and polyadenylation.

As used herein, “a plant-functional enzyme of the nicotinamide adeninedinucleotide salvage synthesis pathway” is an enzyme which whenintroduced into plants, linked to appropriate control elements such asplant expressible promoter and terminator region, can be transcribed andtranslated to yield a enzyme of the NAD salvage synthesis pathwayfunctional in plant cells. Included are the enzymes (and encoding genes)from the NAD salvage synthesis, which are obtained from a yeast orfungus different from Saccharomyces cerevisae.

The latter proteins are very suitable for the methods according to theinvention, since these are less likely to be subject to the enzymaticfeedback regulation etc. to which similar plant-derived enzymes may besubject.

Enzymes involved in the NAD salvage synthesis pathway comprise thefollowing

-   -   Nicotinamidase (EC 3.5.1.19) catalyzing the hydrolysis of the        amide group of nicotinamide, thereby releasing nicotinate and        NH3. The enzyme is also known as nicotinamide deaminase,        nicotinamide amidase, YNDase or nicotinamide amidohydrolase    -   Nicotinate phophoribosyltransferase (EC 2.4.2.11) also known as        niacin ribonucleotidase, nicotinic acid mononucleotide        glycohydrolase; nicotinic acid mononucleotide pyrophosphorylase;        nicotinic acid phosphoribosyltransferase catalyzing the        following reaction        Nicotinate-D-ribonucleotide+diphosphate=nicotinate+5-phospho-α-D-ribose        1-diphosphate    -   Nicotinate-nucleotide adenylyltransferase, (EC 2.7.7.18) also        known as deamido-NAD+pyrophosphorylase; nicotinate        mononucleotide adenylyltransferase; deamindonicotinamide adenine        dinucleotide pyrophsophorylase; NaMT-ATase; nicotinic acid        mononucleotide adenylyltransferase catalyzing the following        reaction        ATP+nicotinate ribonucleotide=diphosphate+deamido-NAD⁺    -   NAD-synthase (EC 6.3.1.5) also known as NAD synthetase;        NAD+synthase; nicotinamide adenine dinucleotide synthetase;        diphosphopyridine nucleotide synthetase, catalyzing the        following reaction        Deamido-NAD⁺+ATP+NH3=AMP+diphosphate+NAD⁺

In one embodiment of the invention, the coding regions encoding thedifferent enzymes of the NAD salvage pathway comprise a nucleotidesequence encoding proteins with the amino acid sequences as set forthhereinafter.

Suitable nucleotide sequences encoding a nicotinamidase similar to PNC1from Saccharomyces cerevisiae but from fungal or yeast-like origininclude a nucleotide sequence encoding a nicotineamidase comprising anamino acid sequence selected from:

the amino acid sequence of accession number XP_(—)444840 (Candidaglabrata)

the amino acid sequence of accession number XP_(—)456073 (Kluyveromyceslactis)

the amino acid sequence of accession number NP_(—)986013 (Eremotheciumgossypii)

the amino acid sequence of accession number XP_(—)888958 (Candidaalbicans)

the amino acid sequence of accession number XP500320 (Yarrowialipolytica)

the amino acid sequence of accession number XP389372 (Giberella zeae)

the amino acid sequence of accession number XP_(—)749509 (Aspergillusfumigatus)

the amino acid sequence of accession number XP_(—)712112 (Candidaalbicans)

the amino acid sequence of accession number BAE56421 (Aspergillusoryzae)

the amino acid sequence of accession number XP_(—)567125 (Cryptococcusneofomans)

the amino acid sequence of accession number XP_(—)964547 (Neurosporacrassa)

the amino acid sequence of accession number XP_(—)712135 (Candidaalbicans)

Suitable nucleotide sequences encoding an NAD(+) synthetase similar toQns1 from Saccharomyces cerevisiae but from fungal origin include anucleotide sequence encoding a NAD(+) synthetase comprising an aminoacid sequence selected from:

the amino acid sequence of accession number XP_(—)448179 (Candidaglabrata)

the amino acid sequence of accession number XP_(—)453643 (Kluyveromyceslactis)

the amino acid sequence of accession number NP_(—)987024 (Eremotheciumgossypii)

the amino acid sequence of accession number XP_(—)500272 (Yarrowialipolytica)

the amino acid sequence of accession number XP_(—)722371 (Candidaalbicans)

the amino acid sequence of accession number XP_(—)456405 (Debaromyceshansenii)

the amino acid sequence of accession number BAE61562 (Aspergillusoryzae)

the amino acid sequence of accession number XP_(—)759702 (Ustilagomaydis)

the amino acid sequence of accession number EAL18079 (Cryptococcusneoformans)

the amino acid sequence of accession number NP_(—)587771(Schizosaccharomyces pombe)

the amino acid sequence of accession number XP_(—)681472 (Aspergillusnidulans)

the amino acid sequence of accession number XP_(—)959191 (Neurosporacrassa)

the amino acid sequence of accession number XP_(—)567726 (Cryptococcusneoformans)

the amino acid sequence of accession number EAQ90706 (Chaetomiumglobosum)

the amino acid sequence of accession number XP_(—)387574 (Giberellazeae)

the amino acid sequence of accession number XP_(—)748008 (Aspergillusfumigatus)

the amino acid sequence of accession number XP_(—)361704 (Magnaporthegrisea)

Suitable nucleotide sequences encoding an Nicotinic acid mononucleotideadenylyltransferase similar to NMA1 from Saccharomyces cerevisiae butfrom fungal origin include a nucleotide sequence encoding a acidmononucleotide adenylyltransferase comprising an amino acid sequenceselected from:

the amino acid sequence of accession number Q06178

the amino acid sequence of accession number XP_(—)444815 (Candidaglabrata)

the amino acid sequence of accession number NP_(—)986687 ((Eremotheciumgossypii)

the amino acid sequence of accession number XP_(—)453005 (Kluyveromyceslactis)

the amino acid sequence of accession number XP_(—)458184(Debaromyceshansenii)

the amino acid sequence of accession number XP_(—)718656 (Candidaalbicans)

the amino acid sequence of accession number XP_(—)504391 (Yarrowialipolytica)

the amino acid sequence of accession number NP_(—)592856(Schizosaccharomyces pombe)

the amino acid sequence of accession number XP_(—)762639 (Ustilagomaydis)

the amino acid sequence of accession number XP_(—)571297 (Cryptococcusneoformans)

the amino acid sequence of accession number BAE57070 (Aspergillusoryzae)

the amino acid sequence of accession number XP_(—)750776 (Aspergillusfumigatus)

the amino acid sequence of accession number XP_(—)659349 (Aspergillusnidulans)

the amino acid sequence of accession number XP_(—)389652 (Giberellazeae)

the amino acid sequence of accession number XP_(—)957634 (Neurosporacrassa)

the amino acid sequence of accession number XP_(—)363364 (Magnaporthegrisea)

the amino acid sequence of accession number XP_(—)758179 (Ustilagomaydis)

the amino acid sequence of accession number EAQ85219 ((Chaetomiumglobosum)

Suitable nucleotide sequences encoding a nicotinatephosphoribosyltransferase similar to NPT1 from Saccharomyces cerevisiaebut from fungal or yeast-like origin include a nucleotide sequenceencoding nicotinate phosphoribosyltransferase comprising an amino acidsequence selected from:

the amino acid sequence of accession number CAA85352 (Saccharomycescerevisae)

the amino acid sequence of accession number XP_(—)448893 (Candidaglabrata)

the amino acid sequence of accession number XP-453357 (Kluyveromyceslactis)

the amino acid sequence of accession number NP_(—)983562 (Eremotheciumgossypii)

the amino acid sequence of accession number XP_(—)462577 (Debaromyceshansenii)

the amino acid sequence of accession number XP_(—)889008 (Candidaalbicans)

the amino acid sequence of accession number XP_(—)500338 (Yarrowialipolytica)

the amino acid sequence of accession number XP_(—)746744 (Aspergillusfumigatus)

the amino acid sequence of accession number BAE64333 (Aspergillusoryzae)

the amino acid sequence of accession number XP_(—)965789 (Neurosporacrassa)

the amino acid sequence of accession number EAQ93453 (Chaetomiumglobosum)

the amino acid sequence of accession number XP_(—)682385 (Aspergillusnidulans)

the amino acid sequence of accession number AAN74808 (Gibberellamoniliformis)

the amino acid sequence of accession number Q9UTK3

the amino acid sequence of accession number XP_(—)361075 (Magnaporthegrisea)

the amino acid sequence of accession number EAL18922 (Cryptococcusneoformans)

the amino acid sequence of accession number XP_(—)568039 (Cryptococcusneoformans)

the amino acid sequence of accession number XP_(—)760597 (Ustilagomaydis)

Suitable nucleotide sequences encoding an Nicotinic acid mononucleotideadenylyltransferase similar to NMA2 from Saccharomyces cerevisiae butfrom fungal or yeast like origin include a nucleotide sequence encodinga acid mononucleotide adenylyltransferase comprising an amino acidsequence selected from:

the amino acid sequence of accession number NP_(—)011524

the amino acid sequence of accession number XP_(—)444815 (Candidaglabrata)

the amino acid sequence of accession number NP_(—)986687 ((Eremotheciumgossypii)

the amino acid sequence of accession number XP_(—)453005 (Kluyveromyceslactis)

the amino acid sequence of accession number XP_(—)458184 (Debaromyceshansenii)

the amino acid sequence of accession number XP_(—)718656 (Candidaalbicans)

the amino acid sequence of accession number XP_(—)504391 (Yarrowialipolytica)

the amino acid sequence of accession number NP_(—)592856(Schizosaccharomyces pombe)

the amino acid sequence of accession number XP_(—)762639 (Ustilagomaydis)

the amino acid sequence of accession number XP_(—)571297 (Cryptococcusneoformans)

the amino acid sequence of accession number BAE57070 (Aspergillusoryzae)

the amino acid sequence of accession number XP_(—)750776 (Aspergillusfumigatus)

the amino acid sequence of accession number XP_(—)659349 (Aspergillusnidulans)

the amino acid sequence of accession number XP_(—)389652 (Giberellazeae)

the amino acid sequence of accession number XP_(—)957634 (Neurosporacrassa)

the amino acid sequence of accession number XP_(—)363364 (Magnaporthegrisea)

the amino acid sequence of accession number XP_(—)758179 (Ustilagomaydis)

the amino acid sequence of accession number EAQ85219 ((Chaetomiumglobosum)

All amino acid sequences referred to by their accession numbers areherein incorporated by reference.

However, it will be clear that variants of these sequences, includinginsertions, deletions and substitutions thereof may be also be used tothe same effect. Variants of the described sequence will have a sequenceidentity which is preferably at least about 80%, or 85 or 90% or 95%with identified sequences of enzymes from the NAD salvage pathway.Preferably, these variants will be functional proteins with the sameenzymatic activity as the enzymes from the NAD salvage pathway. For thepurpose of this invention, the “sequence identity” of two relatednucleotide or amino acid sequences, expressed as a percentage, refers tothe number of positions in the two optimally aligned sequences whichhave identical residues. (×100) divided by the number of positionscompared. A gap, i.e. a position in an alignment where a residue ispresent in one sequence but not in the other, is regarded as a positionwith non-identical residues. The alignment of the two sequences isperformed by the Needleman and Wunsch algorithm (Needleman and Wunsch1970). The computer-assisted sequence alignment above, can beconveniently performed using standard software program such as GAP whichis part of the Wisconsin Package Version 10.1 (Genetics Computer Group,Madison, Wis., USA) using the default scoring matrix with a gap creationpenalty of 50 and a gap extension penalty of 3.

Homologous nucleotide sequence from other fungi or yeast-like organismsmay also be identified and isolated by hybridization under stringentconditions using as probes identified nucleotide sequences encodingenzymes from the NAD salvage pathway.

“Stringent hybridization conditions” as used herein means thathybridization will generally occur if there is at least 95% andpreferably at least 97% sequence identity between the probe and thetarget sequence. Examples of stringent hybridization conditions areovernight incubation in a solution comprising 50% formamide, 5×SSC (150mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6),5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared carrier DNA such as salmon sperm DNA, followed by washing thehybridization support in 0.1×SSC at approximately 65° C., preferablytwice for about 10 minutes. Other hybridization and wash conditions arewell known and are exemplified in Sambrook et al, Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor, N.Y. (1989),particularly chapter 11.

The methods of the invention can be used to obtain plants tolerant todifferent kinds of stress-inducing conditions, particularly abioticstress conditions including submergence, high light conditions, high UVradiation levels, increased hydrogen peroxide levels, droughtconditions, high or low temperatures, increased salinity conditions. Themethods of the invention can also be used to reduce the level of ROS inthe cells of plants growing under adverse conditions, particularlyabiotic stress conditions including submergence, high light conditions,high UV radiation levels, increased hydrogen peroxide levels, droughtconditions, high or low temperatures, increased salinity conditions etc.The level of ROS or the level of NADH can be determined using themethods known in the art, including those described in Example 3.

Using the methods described herein, plants may be obtained wherein thelevel of ROS is equal to or lower than in control plants undernon-stressed conditions, such as but not limited to low light. In theseplants, under non-stressed conditions, the level of ROS may range from50% to 100% of the level of control plants under low light conditions,more particularly from about 60% to about 85%. The level of the ROS inthese plants under stress conditions is about 50% to 80% of the level ofROS in control plants under stress conditions, corresponding to about 60to 80% of the level of ROS in control plants under non-stressedconditions. Similarly, the NADH level in these plants is equal to orhigher than in control plants under non-stressed conditions, such as butnot limited to low light. In these plants, under non-stressedconditions, the level of NADH may range from 100% to 160% of the levelof NADH in control plants under low light conditions, more particularlyfrom about 120% to about 140%. The level of NADH in these plants understress conditions is about 200 to 300% of the level of NADH in controlplants under stress conditions, corresponding to about 100 to 160% ofthe level of ROS in control plants under non-stressed conditions.

Methods to obtain transgenic plants are not deemed critical for thecurrent invention and any transformation method and regenerationsuitable for a particular plant species can be used. Such methods arewell known in the art and include Agrobacterium-mediated transformation,particle gun delivery, microinjection, electroporation of intact cells,polyethyleneglycol-mediated protoplast transformation, electroporationof protoplasts, liposome-mediated transformation, silicon-whiskersmediated transformation etc. The transformed cells obtained in this waymay then be regenerated into mature fertile plants.

The obtained transformed plant can be used in a conventional breedingscheme to produce more transformed plants with the same characteristicsor to introduce the chimeric gene according to the invention in othervarieties of the same or related plant species, or in hybrid plants.Seeds obtained from the transformed plants contain the chimeric genes ofthe invention as a stable genomic insert and are also encompassed by theinvention.

It will be clear that the different stress resistant chimeric genesdescribed herein, with DNA regions encoding different enzymes from theNAD salvage pathway can be combined within one plant cell or plant, tofurther enhance the stress tolerance of the plants comprising thechimeric genes. Thus, in one embodiment of the invention, plant cellsand plants are provided which comprise at least two stress resistantchimeric genes each comprising a different coding region.

The transgenic plant cells and plant lines according to the inventionmay further comprise chimeric genes which will reduce the expression ofendogenous PARP and/or PARG genes as described in WO 00/04173 andPCT/EP2004/003995 These further chimeric genes may be introduced e.g. bycrossing the transgenic plant lines of the current invention withtransgenic plants containing PARP and/or PARG gene expression reducingchimeric genes. Transgenic plant cells or plant lines may also beobtained by introducing or transforming the chimeric genes of theinvention into transgenic plant cells comprising the PARP or PARG geneexpression reducing chimeric genes or vice versa.

For the purpose of the invention, the promoter is a plant-expressiblepromoter. As used herein, the term “plant-expressible promoter” means aDNA sequence which is capable of controlling (initiating) transcriptionin a plant cell. This includes any promoter of plant origin, but alsoany promoter of non-plant origin which is capable of directingtranscription in a plant cell, i.e., certain promoters of viral orbacterial origin such as the CaMV35S (Harpster et al., 1988 Mol. Gen.Genet. 212, 182-190), the subterranean clover virus promoter No 4 or No7 (WO9606932), or T-DNA gene promoters but also tissue-specific ororgan-specific promoters including but not limited to seed-specificpromoters (e.g., WO089/03887), organ-primordia specific promoters (An etal., 1996, The Plant Cell 8, 15-30), stem-specific promoters (Keller etal., 1988, EMBO J. 7, 3625-3633), leaf specific promoters (Hudspeth etal., 1989, Plant Mol Biol 12, 579-589), mesophyl-specific promoters(such as the light-inducible Rubisco promoters), root-specific promoters(Keller et al., 1989, Genes Devel. 3, 1639-1646), tuber-specificpromoters (Keil et al., 1989, EMBO J. 8, 1323-1330), vascular tissuespecific promoters (Peleman et al., 1989, Gene 84, 359-369),stamen-selective promoters (WO 89/10396, WO 92/13956), dehiscence zonespecific promoters (WO 97/13865) and the like.

The chimeric genes of the inventions may also be equipped with a nuclearlocalization signal (“NLS”) functional in plants, operably linked to theDNA region encoding an enzyme of the NAD salvage pathway such as theSV40 NLS.

Having read this document, a person skilled in the art will immediatelyrealize that similar effects with regard to increased stress resistancecan be obtained whenever natural variants of plants are obtained whereinthe endogenous genes coding for NAD salvage pathway enzymes are moreactive or expressed at a higher level. Such variant plants can beobtained by subjecting a population of plants to mutagenesis, such as,but not limited to EMS mutagenesis, followed by a screening for anincreased activity of any one of the NAD salvage pathway enzymes, or acombination thereof.

It will also be immediately clear that a population of differentvarieties or cultivars can be screened for increased tolerance to theabove mentioned stress conditions in general or particular selectedabiotic stresses, followed by a correlation of the increased toleranceto stress conditions with the presence of a particular allele of any ofthe endogenous genes encoding an enzyme of the NAD salvage pathwayenzyme. Such alleles can than be introduced into a plant of interest bycrossing, if the species are sexually compatible, or they may beidentified using conventional techniques as described herein (includinghybridization or PCR amplification) and introduced using recombinant DNAtechnology. Introduction of particularly desired alleles using breedingtechniques may be followed using molecular markers specific for thealleles of interest.

The methods and means described herein are believed to be suitable forall plant cells and plants, both dicotyledonous and monocotyledonousplant cells and plants including but not limited to cotton, Brassicavegetables, oilseed rape, wheat, corn or maize, barley, sunflowers,rice, oats, sugarcane, soybean, vegetables (including chicory, lettuce,tomato), tobacco, potato, sugarbeet, papaya, pineapple, mango,Arabidopsis thaliana, but also plants used in horticulture, floricultureor forestry.

As used herein “comprising” is to be interpreted as specifying thepresence of the stated features, integers, steps or components asreferred to, but does not preclude the presence or addition of one ormore features, integers, steps or components, or groups thereof. Thus,e.g., a nucleic acid or protein comprising a sequence of nucleotides oramino acids, may comprise more nucleotides or amino acids than theactually cited ones, i.e., be embedded in a larger nucleic acid orprotein. A chimeric gene comprising a DNA region which is functionallyor structurally defined, may comprise additional DNA regions etc.

The following non-limiting Examples describe the construction ofchimeric genes to increase stress resistance in plant cells and plantsand the use of such genes.

Unless stated otherwise in the Examples, all recombinant DNA techniquesare carried out according to standard protocols as described in Sambrooket al. (1989) Molecular Cloning: A Laboratory Manual, Second Edition,Cold Spring Harbor Laboratory Press, NY and in Volumes 1 and 2 ofAusubel et al. (1994) Current Protocols in Molecular Biology, CurrentProtocols, USA. Standard materials and methods for plant molecular workare described in Plant Molecular Biology Labfax (1993) by R. D. D. Croy,jointly published by BIOS Scientific Publications Ltd (UK) and BlackwellScientific Publications, UK. Other references for standard molecularbiology techniques include Sambrook and Russell (2001) MolecularCloning: A Laboratory Manual, Third Edition, Cold Spring HarborLaboratory Press, NY, Volumes I and II of Brown (1998) Molecular BiologyLabFax, Second Edition, Academic Press (UK). Standard materials andmethods for polymerase chain reactions can be found in Dieffenbach andDveksler (1995) PCR Primer: A Laboratory Manual, Cold Spring HarborLaboratory Press, and in McPherson at al. (2000) PCR—Basics: FromBackground to Bench, First Edition, Springer Verlag, Germany.

Throughout the specification reference is made to the following entriesin the Sequence listing:

SEQ ID No. 1: XP_(—)444840 (Candida glabrata)

SEQ ID No. 2: XP_(—)456073 (Kluyveromyces lactis)

SEQ ID No. 3: NP_(—)986013 (Eremothecium gossypii)

SEQ ID No. 4: XP_(—)888958 (Candida albicans)

SEQ ID No. 5: XP500320 (Yarrowia lipolytica)

SEQ ID No. 6: XP389372 (Giberella zeae)

SEQ ID No. 7: XP_(—)749509 (Aspergillus fumigatus)

SEQ ID No. 8: XP_(—)712112 (Candida albicans)

SEQ ID No. 9: BAE56421 (Aspergillus oryzae)

SEQ ID No. 10: XP_(—)567125 (Cryptococcus neofomans)

SEQ ID No. 11 XP_(—)964547 (Neurospora crassa)

SEQ ID No. 12: XP_(—)712135 (Candida albicans)

SEQ ID No. 13: XP_(—)448179 (Candida glabrata)

SEQ ID No. 14: XP_(—)453643 (Kluyveromyces lactis)

SEQ ID No. 15: NP_(—)987024 (Eremothecium gossypii)

SEQ ID No. 16: XP_(—)500272 (Yarrowia lipolytica)

SEQ ID No. 17: XP_(—)722371 (Candida albicans)

SEQ ID No. 18: XP_(—)456405 (Debaromyces hansenii)

SEQ ID No. 19: BAE61562 (Aspergillus oryzae)

SEQ ID No. 20: XP_(—)759702 (Ustilago maydis)

SEQ ID No. 21: EAL18079 (Cryptococcus neoformans)

SEQ ID No. 22: NP_(—)587771 (Schizosaccharomyces pombe)

SEQ ID No. 23: XP_(—)681472 (Aspergillus nidulans)

SEQ ID No. 24: XP_(—)959191 (Neurospora crassa)

SEQ ID No. 25: XP_(—)567726 (Cryptococcus neoformans)

SEQ ID No. 26: EAQ90706 (Chaetomium globosum)

SEQ ID No. 27: XP_(—)387574 (Giberella zeae)

SEQ ID No. 28: XP_(—)748008 (Aspergillus fumigatus)

SEQ ID No. 29: XP_(—)361704 (Magnaporthe grisea)

SEQ ID No. 30: Q06178

SEQ ID No. 31: XP_(—)444815 (Candida glabrata)

SEQ ID No. 32: NP_(—)986687 ((Eremothecium gossypii)

SEQ ID No. 33: XP_(—)453005 (Kluyveromyces lactis)

SEQ ID No. 34. XP_(—)458184(Debaromyces hansenii)

SEQ ID No. 35: XP_(—)718656 (Candida albicans)

SEQ ID No. 36: XP_(—)504391 (Yarrowia lipolytica)

SEQ ID No. 37: NP_(—)592856 (Schizosaccharomyces pombe)

SEQ ID No. 38: XP_(—)762639 (Ustilago maydis)

SEQ ID No. 39: XP_(—)571297 (Cryptococcus neoformans)

SEQ ID No. 40: BAE57070 (Aspergillus oryzae)

SEQ ID No. 41: XP_(—)750776 (Aspergillus fumigatus)

SEQ ID No. 42: XP_(—)659349 (Aspergillus nidulans)

SEQ ID No. 43: XP_(—)389652 (Giberella zeae)

SEQ ID No. 44: XP_(—)957634 (Neurospora crassa)

SEQ ID No. 45: XP_(—)363364 (Magnaporthe grisea)

SEQ ID No. 46: XP_(—)758179 (Ustilago maydis)

SEQ ID No. 47: EAQ85219 ((Chaetomium globosum)

SEQ ID No. 48: CAA85352 (Saccharomyces cerevisae)

SEQ ID No. 49: XP_(—)448893 (Candida glabrata)

SEQ ID No. 50: XP_(—)453357 (Kluyveromyces lactis)

SEQ ID No. 51: NP_(—)983562 (Eremothecium gossypii)

SEQ ID No. 52: XP_(—)462577 (Debaromyces hansenii)

SEQ ID No. 53: XP_(—)889008 (Candida albicans)

SEQ ID No. 54: XP_(—)500338 (Yarrowia lipolytica)

SEQ ID No. 55: XP_(—)746744 (Aspergillus fumigatus)

SEQ ID No. 56: BAE64333 (Aspergillus oryzae)

SEQ ID No. 57: XP_(—)965789 (Neurospora crassa)

SEQ ID No. 58: EAQ93453 (Chaetomium globosum)

SEQ ID No. 59: XP_(—)682385 (Aspergillus nidulans)

SEQ ID No. 60: AAN74808 (Gibberella moniliformis)

SEQ ID No. 61: Q9UTK3

SEQ ID No. 62: XP_(—)361075 (Magnaporthe grisea)

SEQ ID No. 63: EAL18922 (Cryptococcus neoformans)

SEQ ID No. 64: XP_(—)568039 (Cryptococcus neoformans)

SEQ ID No. 65: XP_(—)760597 (Ustilago maydis)

SEQ ID No. 66: NP_(—)011524

All amino acid sequences referred to by their accession numbers areherein incorporated by reference.

EXAMPLES Example 1

Assembly of Stress Resistant Chimeric Genes and Introduction into Plants

To increase the stress resistance in plants, a chimeric gene isconstructed using conventional techniques comprising the following DNAfragments in order:

-   -   A promoter region from Cauliflower Mosaic Virus (CaMV 35S);    -   A DNA fragment of about 60 bp corresponding to the untranslated        leader Cab22L;    -   A DNA fragment as mentioned herein elsewhere encoding a NAD        salvage pathway enzyme from fungal or yeast-like origin,        different from PNC1, NMA1, NMA2 or NPT1 from Saccharomyces        cereviseae.    -   A fragment of the 3′ untranslated end from the 35 S transcript        of CaMV (3′ 35S)

This chimeric gene is introduced in a T-DNA vector, between the left andright border sequences from the T-DNA, together with a selectable markergene.

The T-DNA vectors are introduced into Agrobacterium strains comprising ahelper Ti-plasmid using conventional methods. The chimeric genes areintroduced into plants using a conventional transformation method.Transgenic plants exhibit a higher stress resistance than theircounterpart plants without transgenes.

1. A method for obtaining a plant with increased stress resistance, saidmethod comprising a. introducing a chimeric gene into a cell of a plantto obtain a transgenic cell, said chimeric gene comprising the followingoperably linked DNA fragments: i. a plant-expressible promoter; ii. aDNA region coding for a plant-functional nicotinamidase enzyme having atleast 95% identity to SEQ ID NO: 1; iii. a 3′ end region involved intranscription termination and polyadenylation; b. regenerating saidtransgenic cell to obtain a transgenic plant; c. making a population oftransgenic plants comprising the chimeric gene; and d. selecting a plantfrom said population of transgenic plants which exhibits increasedoxidative stress resistance or a reduced level of reactive oxygenspecies, or that maintains a high level of NADH under stress conditionswhen compared to a similar non-transgenic plant.
 2. A chimeric genecomprising the following operably linked DNA fragments: i. aplant-expressible promoter; ii. a DNA region coding for aplant-functional nicotinamidase enzyme having at least 95% identity toSEQ ID NO: 1; and iii. a 3′ end region involved in transcriptiontermination and polyadenylation.
 3. A transgenic plant cell comprisingthe chimeric gene of claim 2, wherein said transgenic plant cellexpresses the plant-functional nicotinamidase enzyme having at least 95%identity to SEQ ID NO:
 1. 4. A transgenic plant comprising the plantcell of claim 3, wherein said transgenic plant expresses theplant-functional nicotinamidase enzyme having at least 95% identity toSEQ ID NO:
 1. 5. The transgenic plant of claim 4, wherein said plant hasa lower level of reactive oxygen species under stress conditions than asimilar plant not comprising the chimeric gene.
 6. A seed comprising achimeric gene comprising the following operably linked DNA fragments: i.a plant-expressible promoter; ii. a DNA region coding for aplant-functional nicotinamidase enzyme having at least 95% identity toSEQ ID NO: 1; and iii. a 3′ end region involved in transcriptiontermination and polyadenylation.
 7. A method to decrease the level ofreactive oxygen species in a plant or a plant cell under stressconditions or to maintain the level of NAD in a plant or plant cellunder stress conditions, said method comprising the step of transformingsaid plant or plant cell with the chimeric gene of claim 2, wherein thetransformation leads to the expression of a plant functionalnicotinamidase enzyme having at least 95% identity to SEQ ID NO: 1.